A current insight into Salmonella's inducible acid resistance.

Salmonella is a diverse and ubiquitous group of bacteria and a major zoonotic pathogen implicated in several foodborne disease outbreaks worldwide. With more than 2500 distinct serotypes, this pathogen has evolved to survive in a wide spectrum of environments and across multiple hosts. The primary and most common source of transmission is through contaminated food or water. Although the main sources have been primarily linked to animal-related food products, outbreaks due to the consumption of contaminated plant-related food products have increased in the last few years. The perceived ability of Salmonella to trigger defensive mechanisms following pre-exposure to sublethal acid conditions, namely acid adaptation, has renewed a decade-long attention. The impact of acid adaptation on the subsequent resistance against lethal factors of the same or multiple stresses has been underscored by multiple studies. Α plethora of studies have been published, aiming to outline the factors that- alone or in combination- can impact this phenomenon and to unravel the complex networking mechanisms underlying its induction. This review aims to provide a current and updated insight into the factors and mechanisms that rule this phenomenon.

The Effect of the PhoP/PhoQ System on the Regulation of Multi-Stress Adaptation Induced by Acid Stress in Salmonella Typhimurium.

Acidic stress in beef cattle slaughtering abattoirs can induce the acid adaptation response of in-plant contaminated Salmonella. This may further lead to multiple resistance responses threatening public health. Therefore, the acid, heat, osmotic and antibiotic resistances of Salmonella typhimurium (ATCC14028) were evaluated after a 90 min adaption in a pH = 5.4 "mild acid" Luria-Bertani medium. Differences in such resistances were also determined between the ∆phoP mutant and wild-type Salmonella strains to confirm the contribution of the PhoP/PhoQ system. The transcriptomic differences between the acid-adapted and ∆phoP strain were compared to explore the role of the PhoP/Q two-component system in regulating multi-stress resistance. Acid adaptation was found to increase the viability of Salmonella to lethal acid, heat and hyperosmotic treatments. In particular, acid adaptation significantly increased the resistance of Salmonella typhimurium to Polymyxin B, and such resistance can last for 21 days when the adapted strain was stored in meat extract medium at 4 °C. Transcriptomics analysis revealed 178 up-regulated and 274 down-regulated genes in the ∆phoP strain. The Salmonella infection, cationic antimicrobial peptide (CAMP) resistance, quorum sensing and two-component system pathways were down-regulated, while the bacterial tricarboxylic acid cycle pathways were up-regulated. Transcriptomics and RT-qPCR analyses revealed that the deletion of the phoP gene resulted in the down-regulation of the expression of genes related to lipid A modification and efflux pumps. These changes in the gene expression result in the change in net negative charge and the mobility of the cell membrane, resulting in enhanced CAMP resistance. The confirmation of multiple stress resistance under acid adaptation and the transcriptomic study in the current study may provide valuable information for the control of multiple stress resistance and meat safety.

bglG Regulates the Heterogeneity Driven by the Acid Tolerance Response in Lacticaseibacillus paracasei L9.

The acid tolerance of lactic acid bacteria is crucial for their fermentation and probiotic functions. Acid adaption significantly enhances the acid tolerance of strains, and the phenotypic heterogeneity driven by the acid tolerance response (ATR) contributes to this process by providing a selective advantage in harsh environments. The mechanism of heterogeneity under the ATR is not yet clear, but individual gene expression differences are recognized as the cause. In this study, we observed four heterogeneous subpopulations (viable, injured, dead, and unstained) of Lacticaseibacillus paracasei L9 (L9) induced by acid adaption (pH 5.0, 40 min) using flow cytometry. The viable subpopulation represented a significantly superior acid tolerance to the injured subpopulation or total population. Different subpopulations were sorted and transcriptomic analysis was performed. Five genes were found to be upregulated in the viable subpopulation and downregulated in the injured subpopulation, and bglG (LPL9_RS14735) was identified as having a key role in this process. Using salicin (glucoside)-inducing gene expression and gene insertion mutagenesis, we verified that bglG regulated the heterogeneity of the acid stress response and that the relevant mechanisms might be related to activating hsp20. This study provides new evidence for the mechanism of the ATR and may contribute to the theoretical basis of improving the acid tolerance of Lacticaseibacillus paracasei L9.

A novel RofA-family transcriptional regulator, GadR, controls the development of acid resistance in Listeria monocytogenes.

Stomach acid provides a significant innate barrier to the entry of the food-borne pathogen Listeria monocytogenes into the human gastrointestinal tract. A key determinant of acid resistance in this bacterium is the conserved glutamate decarboxylase system, GadD2 (encoded by the gadT2D2 operon), which helps to maintain the intracellular pH during exposure to gastric acid. In this study, we identified a premature stop codon in a gene located immediately downstream of the gadT2D2 operon that was highly linked to an acid-sensitive phenotype. When this open reading frame was restored through homologous recombination, an acid-resistant phenotype was restored. Through a series of genetic, transcriptomic, and survival experiments, we established that this gene, which we designated gadR, encodes a transcriptional regulator of the gadT2D2 operon. GadR belongs to the RofA family of regulators, primarily found in streptococci, where they are involved in regulating virulence. The data further showed that gadR plays a critical role in the development of acid resistance in response to mild acid exposure, a response that is known as the adaptive acid tolerance response (ATR). A deletion analysis of the gadT2D2 promoter region identified two 18-bp palindromic sequences that are required for the GadR-mediated induction of gadT2D2, suggesting that they act as binding sites for GadR. Overall, this study uncovers a new RofA-like regulator of acid resistance in L. monocytogenes, which plays a significant role in both growth phase-dependent acid resistance and ATR and accounts for previously observed strain-to-strain differences in survival at low pH.IMPORTANCEThe ability to survive the acidic conditions found in the stomach is crucial for the food-borne pathogen Listeria monocytogenes to gain access to the mammalian gastrointestinal tract. Little is currently known about how acid resistance is regulated in this pathogen and why this trait is highly variable between strains. Here, we used comparative genomics to identify a novel RofA-family transcriptional regulator, GadR, that controls the development of acid resistance. The RofA family of regulators was previously found only in a small group of bacterial pathogens, including streptococci, where they regulate virulence properties. We show that gadR encodes the dominant regulator of acid resistance in L. monocytogenes and that its sequence variability accounts for previously observed differences between strains in this trait. Together, these findings significantly advance our understanding of how this important pathogen copes with acid stress and suggest a potential molecular target to aid its control in the food chain.

The role of PhoP/PhoQ system in regulating stress adaptation response in Escherichia coli O157:H7.

The development of acid tolerance response (ATR) as a result of low pH in Escherichia coli O157:H7 (E. coli O157:H7) contaminating beef during processing is considered a major food safety concern. Thus, in order to explore the formation and molecular mechanisms of the tolerance response of E. coli O157:H7 in a simulated beef processing environment, the resistance of a wild-type (WT) strain and its corresponding ΔphoP mutant to acid, heat, and osmotic pressure was evaluated. Strains were pre-adapted under different conditions of pH (5.4 and 7.0), temperature (37 °C and 10 °C), and culture medium (meat extract and Luria-Bertani broth media). In addition, the expression of genes related to stress response and virulence was also investigated among WT and ΔphoP strains under the tested conditions. Pre-acid adaptation increased the resistance of E. coli O157:H7 to acid and heat treatment while resistance to osmotic pressure decreased. Moreover, acid adaptation in meat extract medium simulating slaughter environment increased ATR, whereas pre-adaptation at 10 °C reduced the ATR. Furthermore, it was shown that mildly acidic conditions (pH = 5.4) and the PhoP/PhoQ two-component system (TCS) acted synergistically to enhance acid and heat tolerance in E. coli O157:H7. Additionally, the expression of genes related to arginine and lysine metabolism, heat shock, and invasiveness was up-regulated, which revealed that the mechanism of acid resistance and cross-protection under mildly acidic conditions was mediated by the PhoP/PhoQ TCS. Both acid adaptation and phoP gene knockout reduced the relative expression of stx1 and stx2 genes which were considered as critical pathogenic factors. Collectively, the current findings indicated that ATR could occur in E. coli O157:H7 during beef processing. Thus, there is an increased food safety risk due to the persistence of tolerance response in the following processing conditions. The present study provides a more comprehensive basis for the effective application of hurdle technology in beef processing.

The acid tolerance responses of the Salmonella strains isolated from beef processing plants.

The development of the stationary-phase, low-pH-inducible acid tolerance response (ATR) in the Salmonella contaminant of beef during the processing arises food safety concerns, because it may evoke bacterial coping mechanisms against bactericidal insults and alter gene expression that contribute to pathogen virulence. However, information on the development of the ATR and the stability (defined as the capacity to maintain the acquired acid tolerance after induction) in the Salmonella during the production and distribution of beef is limited. After adaptation overnight, ATRs in the 79 strains of Salmonella isolated from beef processing plants were investigated by comparing the log reduction in the 2-h acid challenge trials at pH 3.0. Six representative strains were selected to further estimate the effect of three factors in the incubation period on the development of the ATR, including adapted pH values (5.0, 5.4, 6.0, and 7.0), temperatures (10 °C and 37 °C), and the adaptation media (meat extract and brain heart infusion media). The stability of acid tolerance during the long-time chilled storage (4 °C for 13 days) was also observed on two strains of serotypes S. Derby and S. Meleagridis. All the strains isolated from beef processing plants exhibited an enhanced acid tolerance indicating the widespread existence of ATR. The results also revealed that strain variability was present in the development of ATR. Significant tolerance to lethal acidic environments (pH 3.0) was found when the Salmonella strains had been acid-adapted in meat extract at pH 5.0, pH 5.4, or pH 6.0, which indicated the possible induction of ATR during beef production. After the acid adaptations, the population reduction after the acid challenge (BHI, pH = 3) in the strains was significantly lower than the non-induced at the 1d, 7 day and 13 day's storage in meat extract media at 4 °C, which revealed the persistence of ATR during beef distribution. Compared to 37 °C, adaptation in lower temperature (10 °C) significantly reduced the ATR and no ATR was developed when adapted in 4 °C. This emphasizes the importance of keeping a low temperature of beef throughout the supply chains of beef industry.

Pyruvate Oxidase as a Key Determinant of Pneumococcal Viability during Transcytosis across Brain Endothelium.

Streptococcus pneumoniae invades a myriad of host tissues following efficient breaching of cellular barriers. However, strategies adopted by pneumococcus for evasion of host intracellular defenses governing successful transcytosis across host cellular barriers remain elusive. In this study, using brain endothelium as a model host barrier, we observed that pneumococcus containing endocytic vacuoles (PCVs), formed following S. pneumoniae internalization into brain microvascular endothelial cells (BMECs), undergo early maturation and acidification, with a major subset acquiring lysosome-like characteristics. Exploration of measures that would preserve pneumococcal viability in the lethal acidic pH of these lysosome-like vacuoles revealed a critical role of the two-component system response regulator, CiaR, which was previously implicated in induction of acid tolerance response. Pyruvate oxidase (SpxB), a key sugar-metabolizing enzyme that catalyzes oxidative decarboxylation of pyruvate to acetyl phosphate, was found to contribute to acid stress tolerance, presumably via acetyl phosphate-mediated phosphorylation and activation of CiaR, independent of its cognate kinase CiaH. Hydrogen peroxide, the by-product of an SpxB-catalyzed reaction, was also found to improve pneumococcal intracellular survival by oxidative inactivation of lysosomal cysteine cathepsins, thus compromising the degradative capacity of the host lysosomes. As expected, a ΔspxB mutant was found to be significantly attenuated in its ability to survive inside the BMEC endocytic vacuoles, reflecting its reduced transcytosis ability. Collectively, our studies establish SpxB as an important virulence determinant facilitating pneumococcal survival inside host cells, ensuring successful trafficking across host cellular barriers. IMPORTANCE Host cellular barriers have innate immune defenses to restrict microbial passage into sterile compartments. Here, by focusing on the blood-brain barrier endothelium, we investigated mechanisms that enable Streptococcus pneumoniae to traverse through host barriers. Pyruvate oxidase, a pneumococcal sugar-metabolizing enzyme, was found to play a crucial role in this via generation of acetyl phosphate and hydrogen peroxide. A two-pronged approach consisting of acetyl phosphate-mediated activation of acid tolerance response and hydrogen peroxide-mediated inactivation of lysosomal enzymes enabled pneumococci to maintain viability inside the degradative vacuoles of the brain endothelium for successful transcytosis across the barrier. Thus, pyruvate oxidase is a key virulence determinant and can potentially serve as a viable candidate for therapeutic interventions for better management of invasive pneumococcal diseases.

The responses of Salmonella enterica serovar Typhimurium to vanillin in apple juice through global transcriptomics.

Salmonella enterica serovar Typhimurium can survive some extreme environment in food processing, and vanillin generally recognized as safe is bactericidal to pathogens. Thus, we need to explore the responses of S. Typhimurium to vanillin in order to apply this antimicrobial agent in food processing. In this study, we exposed S. Typhimurium to commercial apple juice with/without vanillin (3.2 mg/mL) at 45 °C for 75 min to determine the survival rate. Subsequently, the 10-min cultures were selected for transcriptomic analysis. Using high-throughput RNA sequencing, genes related to vanillin resistance and their expression changes of S. Typhimurium were identified. The survival curve showed that S. Typhimurium treated with vanillin were inactivated by 5.5 log after 75 min, while the control group only decreased by 2.3 log. Such a discrepancy showed the significant antibacterial effect of vanillin on S. Typhimurium. As a result, 265 differentially expressed genes (DEGs) were found when coping with vanillin, among which, 225 showed up-regulation and 40 DEGs were down-regulated. Treated with vanillin, S. Typhimurium significantly up-regulated genes involved in cell membrane, acid tolerance response (ATR) and oxidative stress response, cold shock cross-protection, DNA repair, virulence factors and some key regulators. Firstly, membrane-related genes, including outer membrane (bamE, mepS, ygdI, lolB), inner membrane (yaiY, yicS) and other proteins (yciC, yjcH), were significantly up-regulated because of the damaged cell membrane. Then, up-regulated proteins associated with arginine synthesis (ArgABCDIG) and inward transportation (ArtI, ArtJ, ArtP and HisP), participated in ATR to pump out the protons inside the cell in this scenario. Next, superoxide stress response triggered by vanillin was found to have a significant up-regulation as well, which was controlled by SoxRS regulon. Besides, NADH-associated (nuoA, nuoB, nuoK, nadE, fre and STM3021), thioredoxin (trxA, trxC, tpx and bcp) and glutaredoxin (grxC and grxD) DEGs led to the increase of the oxidative stress response. Cold shock proteins such as CspA and CspC showed an up-regulation, suggesting it might play a role in cross-protecting S. Typhimurium from vanillin stress. Furthermore, DEGs in DNA repair and virulence factors, including flagellar assembly, adhesins and type III secretion system were up-regulated. Some regulators like fur, rpoE and csrA played a pivotal role in response to the stress caused by vanillin. Therefore, this study sounds an alarm for the risks caused by stress tolerance of S. Typhimurium in food industry.

Vibrio parahaemolyticus CadC regulates acid tolerance response to enhance bacterial motility and cytotoxicity.

Pathogens adapted to sub-lethal acidic conditions could increase the virulence and survival ability under lethal conditions. In the aquaculture industry, feed acidifiers have been used to increase the growth of aquatic animals. However, there is limited study on the effects of acidic condition on the virulence and survival of pathogens in aquaculture. In this study, we investigated the survival ability of Vibrio parahaemolyticus at lethal acidic pH (4.0) after adapted the bacteria to sub-lethal acidic pH (5.5) for 1 hr. Our results indicated that the adapted strain increased the survival ability at lethal acidic pH invoked by an inorganic (HCl) or organic (citric) acid. RNA-sequencing (RNA-seq) results revealed that 321 genes were differentially expressed at the sub-lethal acidic pH including cadC, cadBA and groES/groEL relating to acid tolerance response (ATR), as well as genes relating to outer membrane, heat-shock proteins, phosphotransferase system and flagella system. Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed that cadC and cadBA were upregulated under sub-lethal acidic conditions. The CadC protein could directly regulate the expression of cadBA to modulate the ATR in V. parahaemolyticus. RNA-seq data also indicated that 113 genes in the CadC-dependent way and 208 genes in the CadC-independent way were differentially expressed, which were related to the regulation of ATR. Finally, the motility and cytotoxicity of the sub-lethal acidic adapted wild type (WT) were significantly increased compared with the unadapted strain. Our results demonstrated that the dietary acidifiers may increase the virulence and survival of V. parahaemolyticus in aquaculture.

Acid tolerance in early colonizers of oral biofilms.

BACKGROUND: In caries, low pH drives selection and enrichment of acidogenic and aciduric bacteria in oral biofilms, and development of acid tolerance in early colonizers is thought to play a key role in this shift. Since previous studies have focussed on planktonic cells, the effect of biofilm growth as well as the role of a salivary pellicle on this process is largely unknown. We explored acid tolerance and acid tolerance response (ATR) induction in biofilm cells of both clinical and laboratory strains of three oral streptococcal species (Streptococcus gordonii, Streptococcus oralis and Streptococcus mutans) as well as two oral species of Actinomyces (A. naeslundii and A. odontolyticus) and examined the role of salivary proteins in acid tolerance development. METHODS: Biofilms were formed on surfaces in Ibidi® mini flow cells with or without a coating of salivary proteins and acid tolerance assessed by exposing them to a challenge known to kill non-acid tolerant cells (pH 3.5 for 30 min) followed by staining with LIVE/DEAD BacLight and confocal scanning laser microscopy. The ability to induce an ATR was assessed by exposing the biofilms to an adaptation pH (pH 5.5) for 2 hours prior to the low pH challenge. RESULTS: Biofilm formation significantly increased acid tolerance in all the clinical streptococcal strains (P < 0.05) whereas the laboratory strains varied in their response. In biofilms, S. oralis was much more acid tolerant than S. gordonii or S. mutans. A. naeslundii showed a significant increase in acid tolerance in biofilms compared to planktonic cells (P < 0.001) which was not seen for A. odontolyticus. All strains except S. oralis induced an ATR after pre-exposure to pH 5.5 (P < 0.05). The presence of a salivary pellicle enhanced both acid tolerance development and ATR induction in S. gordonii biofilms (P < 0.05) but did not affect the other bacteria to the same extent. CONCLUSIONS: These findings suggest that factors such as surface contact, the presence of a salivary pellicle and sensing of environmental pH can contribute to the development of high levels of acid tolerance amongst early colonizers in oral biofilms which may be important in the initiation of caries.

Acid tolerance response of Salmonella during simulated chilled beef storage and its regulatory mechanism based on the PhoP/Q system.

To investigate the persistence of acid tolerance response (ATR) and the regulatory mechanism during chilled storage, Salmonella ATCC 14028 and the △phoP mutant were acid adapted and then incubated in meat extract at 4 °C for 24 days as simulated beef storage. The bacterial population, D values and expression of PhoP/PhoQ linked genes of both strains were determined at 6-day intervals. Although a mild suppression effect on the D values of adapted Salmonella was found during the long-time storage in meat extract at 4 °C, the D value of adapted strains was significantly higher than non-adapted strains, indicating the persistence of ATR during the whole aging and distribution of beef posing a threat to food safety. The fact that low temperature inhibits the formation of ATR at the early adapted stage emphasizes the importance of keeping a low-temperature environment during slaughter. An interaction between the acidic adaptation and phoP gene on D values was found and the expression levels of adiA, adiY, cadA and cadB genes was significantly reduced in the △phoP mutant, suggesting that PhoP/Q system plays an important role in the ATR by sensing the pH and regulating lysine and arginine decarboxylation directly or indirectly.

Physiological and Transcriptional Responses of Streptomyces albulus to Acid Stress in the Biosynthesis of ε-Poly-L-lysine.

Streptomyces albulus has commercially been used for the production of ε-poly-L-lysine (ε-PL), a natural food preservative, where acid stress is inevitably encountered in the biosynthesis process. To elucidate the acid tolerance response (ATR), a comparative physiology and transcriptomic analysis of S. albulus M-Z18 at different environmental pH (5.0, 4.0, and 3.0) was carried out. In response to acid stress, cell envelope regulated the membrane fatty acid composition and chain length to reduce damage. Moreover, intracellular pH homeostasis was maintained by increasing H+-ATPase activity and intracellular ATP and amino acid (mainly arginine, glutamate, aspartate and lysine) concentrations. Transcriptional analysis based on RNA-sequencing indicated that acid stress aroused global changes and the differentially expressed genes involved in transcriptional regulation, stress-response protein, transporter, cell envelope, secondary metabolite biosynthesis, DNA and RNA metabolism and ribosome subunit. Consequently, the ATR of S. albulus was preliminarily proposed. Notably, it is indicated that the biosynthesis of ε-PL is also a response mechanism for S. albulus to combat acid stress. These results provide new insights into the ATR of S. albulus and will contribute to the production of ε-PL via adaptive evolution or metabolic engineering.

Tolerance to acid and alkali by Streptococcus infantarius subsp. infantarius strain 25124 isolated from fermented nixtamal dough: Pozol. Studies in APT broth.

Pozol is a beverage prepared with maize dough made after boiling the kernels in limewater. This pretreatment could act as a selective force that shapes the starter microbiota, with microorganisms able to survive the fermentation. Since Streptococcus infantarius subsp. infantarius (Sii) dominates in pozol, we evaluated the effect of acid and alkali stresses on strain Sii-25124 in commercial APT broth as a first attempt to assess its adaptation capacity. Results suggest that Sii-25124 has adaptative advantages to pH changes that possibly contribute to its persistence even after the acidification of the dough. Its cardinal pH values were 4.0 and 11.0, with an optimum between 6.6 and 8.0. It showed alkali tolerance unlike other pozol Sii strains. Adaptation at pH 4.0, 10.0 and 11.0, compared with non-adapted cells, induced acid tolerance enhancing survival at pH 3.6 (P < 0.05); a 2 min heat shock at 62 °C induced alkali tolerance response enhancing survival at pH 10.5 (P < 0.05). The up-regulation of dnaK, groEL, ptsG and atpB was observed during 5 h of exposition at pH 3.6, 4.0 and 10.0, showing similar expression rates after induction by acid shock or alkaline stress. Changes of atpB were more evident having almost five-fold induction during long-term stress.

Global transcriptomic analysis of Cronobacter sakazakii CICC 21544 by RNA-seq under inorganic acid and organic acid stresses.

Cronobacter sakazakii is a common foodborne pathogen that can tolerate various stress conditions. Acidic environment is a common stress condition encountered by bacteria in food processing and gastrointestinal digestion, including both inorganic and organic acids. In order to elucidate the Acid Tolerance Response (ATR) of C. sakazakii, we performed high-throughput RNA-seq to compare gene expression under hydrochloric acid and citric acid stresses. In this study, 107 differentially expressed genes (DEGs) were identified in both acids, of which 85 DEGs were functionally related to the regulation of acid tolerance. Multiple layers of mechanisms may be applied by C. sakazakii in response to acid stress: Firstly, in order to reduce excessive intracellular protons, C. sakazakii pumps them out through trans-membrane proteins or consumes them through metabolic reactions. Secondly, under acidic conditions, a large amount of reactive oxygen species and hydroxyl radicals accumulate in the cells, resulting in oxidative damage. C. sakazakii protects cells by up-regulating the antioxidant stress genes such as soxS and madB. Thirdly, C. sakazakii chooses energy efficient metabolic pathways to reduce energy consumption and maintain necessary processes. Finally, genes involved in chemotaxis and motility were differentially expressed to respond to different acidic conditions. This study systematically analyzed the acid-resistant mechanism of C. sakazakii under the stress of organic and inorganic acids, and provided a theoretical basis for better control of its contamination in food.

Streptococcus mutans isolated from children with severe-early childhood caries form higher levels of persisters.

OBJECTIVES: Dental caries is the most common chronic infectious disease in children. Streptococcus mutans, the main cariogenic bacterial species, produces persisters, nongrowing dormant variants of regular cells associated with chronicity of diseases. We hypothesized that the recurrent nature of caries, particularly within populations with high-caries risk, is due partly to specific phenotypic features of S. mutans such as its ability to form persisters. We aimed to investigate the genotypic and phenotypic differences between the S. mutans from children with severe early-childhood caries (S-ECC) and those without caries. METHODS: S. mutans from plaque samples of caries-free (CF) and S-ECC children were tested for their ability to adapt to a lethal pH in an acid tolerance response assay. The persister levels of S. mutans isolates was quantified in both groups. RESULTS: S. mutanswas identified in all 23 S-ECC but only 6 of the 21 CF subjects. In most subjects, only one dominant S. mutans genotype was detected. No statistically significant differences in the mean survival percentage of S. mutans were observed between the two groups at a lethal pH of 3.5. However, the dominant genotype within a particular S-ECC subject exhibited a higher percentage of cell survival compared to those in the CF group. In S-ECC patients, S. mutans isolates displayed a ∼15-fold higher persistence phenotype than S. mutans isolates from CF patients. CONCLUSIONS: The ability of S. mutans to produce high levels of persisters may contribute to part of an individual's ability to control caries disease activity and recurrent lesions.

Prior Exposure to Dry-Cured Meat Promotes Resistance to Simulated Gastric Fluid in Salmonella Typhimurium.

This study assessed if exposure of foodborne Salmonella enterica in Brazilian dry-cured loin (BDL) affects pathogen inactivation in simulated gastric fluid (SGF). The acid tolerance responses of three Salmonella enterica serovars, Typhimurium, Derby and Panama, were assessed by an acid challenge trial at pH 3.0 for 4 h following pre-adaptation to three conditions: neutral pH, acidic pH (4.5) or BDL matrix. The influence of Salmonella exposure temperature and time in the BDL on pathogen gastric fluid resistance was evaluated by the response surface methodology. The Salmonella serovars acquired acid tolerance when exposed to the BDL matrix and their response to acid stress was strain-dependent, with S. Typhimurium being the most tolerant strain. S. Typhimuirum exposed to temperatures >25 °C in the BDL matrix displayed increased resistance to SGF. By using the response surface methodology, it was determined that S. Typhimurium becomes less resistant against SGF if maintained in the BDL matrix at temperatures <7 °C, reinforcing the recommendation to store dry-cured meat under refrigeration in order to minimize consumer risks. The results presented herein point to a novel aspect of hurdle technology that should be taken into account to further understand the risks associated with hurdle-stable meat product, such as dry-cured meats, concerning foodborne pathogen contamination.

Transcriptional analysis reveals the relativity of acid tolerance and antimicrobial peptide resistance of Salmonella.

The objective of this study was to comprehensively identify the target genes induced by acid stimulation in Salmonella, and to clarify the relativity of acid tolerance and antimicrobial peptide resistance. A clinical S. Typhimurium strain, S6, was selected and performed a transcriptome analysis under the acid tolerance response. In total, we found 1461 genes to be differentially expressed, including 721 up-regulated and 740 down-regulated genes. Functional annotation revealed differentially expressed genes to be associated with regulation, metabolism, transport, virulence, and motility. Interestingly, KEGG pathway analysis demonstrated that the induced genes by acid were enriched in cationic antimicrobial peptide resistance, sulfur relay system, ABC transporters, and two-component system pathway. Therein, PhoQ belonging to the two-component system PhoP-PhoQ that promotes virulence by detecting the macrophage phagosome and controls the transcript levels of many genes associated with the resistance to AMPs; MarA, a multiple antibiotic resistance factor; SapA, one of the encoding gene of sapABCDF operon that confers resistance to small cationic peptides of Salmonella; YejB, one of the encoding gene of yejABEF operon that confers resistance to antimicrobial peptides and contributes to the virulence of Salmonella, were all induced by acid stimulation, and could potentially explain that there is a correlation between acid tolerance and AMPs resistance, and finally affects the virulence of intracellular pathogenic bacteria.

Physiological, Structural, and Functional Analysis of the Paralogous Cation-Proton Antiporters of NhaP Type from Vibrio cholerae.

The transmembrane K+/H+ antiporters of NhaP type of Vibrio cholerae (Vc-NhaP1, 2, and 3) are critical for maintenance of K+ homeostasis in the cytoplasm. The entire functional NhaP group is indispensable for the survival of V. cholerae at low pHs suggesting their possible role in the acid tolerance response (ATR) of V. cholerae. Our findings suggest that the Vc-NhaP123 group, and especially its major component, Vc-NhaP2, might be a promising target for the development of novel antimicrobials by narrowly targeting V. cholerae and other NhaP-expressing pathogens. On the basis of Vc-NhaP2 in silico structure modeling, Molecular Dynamics Simulations, and extensive mutagenesis studies, we suggest that the ion-motive module of Vc-NhaP2 is comprised of two functional regions: (i) a putative cation-binding pocket that is formed by antiparallel unfolded regions of two transmembrane segments (TMSs V/XII) crossing each other in the middle of the membrane, known as the NhaA fold; and (ii) a cluster of amino acids determining the ion selectivity.

Free lactic acid production under acidic conditions by lactic acid bacteria strains: challenges and future prospects.

Lactic acid (LA) is an important platform chemical due to its significant applications in various fields and its use as a monomer for the production of biodegradable poly(lactic acid) (PLA). Free LA production is required to get rid of CaSO4, a waste material produced during fermentation at neutral pH which will lead to easy purification of LA required for the production of biodegradable PLA. Additionally, there is no need to use corrosive acids to release free LA from the calcium lactate produced during neutral fermentation. To date, several attempts have been made to improve the acid tolerance of lactic acid bacteria (LAB) by using both genome-shuffling approaches and rational design based on known mechanisms of LA tolerance and gene deletion in yeast strains. However, the lack of knowledge and the complexity of acid-tolerance mechanisms have made it challenging to generate LA-tolerant strains by simply modifying few target genes. Currently, adaptive evolution has proven an efficient strategy to improve the LA tolerance of individual/engineered strains. The main objectives of this article are to summarize the conventional biotechnological LA fermentation processes to date, assess their overall economic and environmental cost, and to introduce modern LA fermentation strategies for free LA production. In this review, we provide a broad overview of free LA fermentation processes using robust LAB that can ferment in acidic environments, the obstacles to these processes and their possible solutions, and the impact on future development of free LA fermentation processes commercially.

In vivo repressed genes of Vibrio cholerae reveal inverse requirements of an H+/Cl- transporter along the gastrointestinal passage.

The facultative human pathogen Vibrio cholerae changes its transcriptional profile upon oral ingestion by the host to facilitate survival and colonization fitness. Here, we used a modified version of recombination-based in vivo expression technology to investigate gene silencing during the in vivo passage, which has been understudied. Using a murine model of cholera, we screened a V. cholerae transposon library composed of 10,000 randomly generated reporter fusions and identified 101 in vivo repressed (ivr) genes. Our data indicate that constitutive expression of ivr genes reduces colonization fitness, highlighting the necessity to down-regulate these genes in vivo. For example, the ivr gene clcA, encoding an H+/Cl- transporter, could be linked to the acid tolerance response against hydrochloric acid. In a chloride-dependent manner, ClcA facilitates survival under low pH (e.g., the stomach), but its presence becomes detrimental under alkaline conditions (e.g., lower gastrointestinal tract). This pH-dependent clcA expression is controlled by the LysR-type activator AphB, which acts in concert with AphA to initiate the virulence cascade in V. cholerae after oral ingestion. Thus, transcriptional networks dictating induction of virulence factors and the repression of ivr genes overlap to regulate in vivo colonization dynamics. Overall, the results presented herein highlight the impact of spatiotemporal gene silencing in vivo. The molecular characterization of the underlying mechanisms can provide important insights into in vivo physiology and virulence network regulation.